4. Desarrollo por acumulación por desposesión, resistencias campesinas y educación
4.2. Educación comunitaria y construcción de planes de vida desde los desca
5. Summary
Gene therapy poses a relatively new approach for treatment of various diseases (first gene therapy clinical trial in 1990). It is still on an experimental basis and there will be still research needed before the first gene therapy product will be accepted for commercial sale. The major hurdles for reaching this so far have been safety and efficiency, two problems generally associated with viral and nonviral gene therapy, respectively. To avoid the safety issues arising from viral delivery, such as reported insertional mutagenesis and severe immune responses, lately the focus has been turned to nonviral vectors. To deal with the low efficiency of these vectors much work has been done on the gene transfer vehicle of the DNA cargo, i.e. liposome and polyplex formulations, for example. But also the construction of the DNA itself, the plasmid, can influence the gene therapy potential considerably, as reported in this thesis.
By choosing and combining the modules of the plasmids in an optimised way, transgene expression can both be enhanced and prolonged. This has been shown by combining a ubiquitous EF-1 promoter with a CMV enhancer, the latter originating either from murine or human CMV. The plasmid with the human CMV enhancer showed clearly a higher and longer-lasting transgene expression in vivo in comparison with the murine analogue, probably due to a better promoter-enhancer interaction. This advantage in expression was also paired with a higher plasmid copy number, showing that a high expression of a plasmid leads to a better retention thereof in the host cell, an important factor for gene therapy. In all cases with plasmids containing mCMV or hCMV enhancers, the transgene signal is first constant, but drops abruptly about 2 weeks after application. To clarify the reason for this sudden reduction of signal the experiment was repeated in immunodeficient mice. The result showed, in contrast to previous measurements, stable expression signals, indicating that the immune system was responsible for the loss of transgene signal. We could also show that a high level of transgene expression was necessary to trigger such an immune response: A plasmid with a CMV promoter showed similar expression profiles in both immunocompetent and –deficient mice; a sharp decrease from day one after application, which within a few days after application stabilised on much lower levels, but showed no further decrease 2 weeks after application. The early signal drop is probably due to previously reported methylation of the CMV
promoter and leads to lower transgene signals, which do not elicit a strong immune response.
The strong, viral CMV promoter suffers from early silencing processes, whereas the signal from the mammalian EF-1 promoter is more stable. With the idea of taking advantage of the best features of the two, a new synthetic promoter was designed by combining the shared homologous regions. The novel promoter, SCEP, showed enhanced transgene signals in vivo compared to both the CMV and EF-1 promoter, but elicited, because of the high levels of transgene product, in the same fashion as the EF-1 containing promoter, an immune response, which led to a drop in signal two weeks after plasmid injection. The two luciferase variants used here as reporter genes are apparently mitogenic, thus, reduces the longevity of their expression. Therefore, it would be of great interest to investigate other transgenes, i.e. endogenous therapeutic genes with less or no immunological effect, to evaluate if the stability of the gene expression can be increased and to which extent.
The transgene signal durability is also connected with the cycling frequency of the transfected cells, as shown in a slow-growing tumour model. The plasmids were introduced into the tumours by electroporation and the tumour cells were isolated and taken into cell culture directly or two weeks after electroporation. Measurements of the transgene signal revealed constant levels in the tumours over one month time, whereas the signal in the cell culture, where the cell doubling time is considerably shorter, was lost within 2 weeks. This result points to the event of mitosis as a decisive time point when plasmids easily are lost, and should be taken into consideration when choosing the target cells for gene therapy.
One way of reducing plasmid loss caused by the host immune defence is to minimise the amount of unmethylated CpG´s in the plasmid sequence. Reduction of the CpG´s can be done either by an overall elimination at the step of plasmid design, or later in the plasmid production process by removing the bacterial backbone, containing most of the unmethylated CpG´s. Thus, a minicircle is formed, containing only the mammalian expression cassette. By a highly efficient, novel production process one model minicircle was produced and in this work tested biologically. The minicircle showed an advantage against the corresponding full-length plasmid in higher application concentrations in vivo. The approach of minicircles is therefore a promising part of the puzzle to complete an efficient therapeutic vector, especially since its production now is efficient enough to be scaled up.
5 Summary 62
One last area of plasmid regulation design evaluated in this work concerned the transcriptional control using liver specific promoters. Compared to the CMV promoter these liver specific promoters were generally weaker, but the transcriptional strength could be enhanced by combination with a hCMV enhancer. The strongest of the tested hybrid liver promoter/ hCMV enhancer was based on the AFP promoter, which is silent in adult liver tissue and reactivated in most HCC. It was tested in vitro and the result showed a low transgene expression in off-target cell lines and high in HCC cell lines, a feature which can be used for transcriptional targeting of cancer cells in the treatment of HCC.
6. Appendix
6.1.
Abbreviations
AFP α-fetoproteinAMP adenosine monophosphate Amp ampicillin resistance gene AP-1, AP-2 activating protein 1, 2 APC antigen presenting cell ATF activating transcription factor BCG bacille Calmette Guérin bp base pair
BRE TFIIB recognition element BSA bovine serum albumin
c/p-ratio Weight ratio of conjugate to plasmid cAMP cyclic adenine monophosphate cDNA complementary DNA
CMV Cytomegalovirus
CMV-IEP cytomegalovirus immediate early promoter CpG cytosine-guanine dinucleotide
CRE cyclic AMP response element CREB cAMP response element binding
CTF CAAT (controlled amino acid treatment) transcription factor DC dendritic cell
DMEM Dulbecco´s modified Eagle´s medium DMSO Dimethyl sulfoxide
DNA deoxyribonucleic acid
DPE down-stream promoter elements DTT Dithiothreitol
E. coli Escherichia coli
EBV Epstein-Barr virus EC endothelial cell
6 Appendix 64
EDTA ethylenediaminetetraacetic acid EF-1 elongation factor-1α
eFLuc enhanced firefly luciferase
EGFP enhanced green fluorescent protein ER endoplasmic reticulum
EtBr Ethidium bromide FCS Fetal calf serum Fig. figure
GAPDH Glyceraldehyde-3-phosphate dehydrogenase GFAP glial fibrillary acidic protein
GLuc Gaussia princeps luciferase HBS HEPES-buffered saline HBV hepatitis B virus
HCC hepatocellular carcinoma hCMV, mCMV human, murine cytomegalovirus HDAC histone deacetylase
HEPES N-(2-hydroxyethyl) piperazine-N’-(2-ethansulfonic acid) hK2 human glandular kallikrein 2
HSV tk herpes simplex virus thymidine kinase gene i.p. intraperitoneal
i.t. intratumoral i.v. intravenous IFN interferon
IKK IκB-kinase complex IL interleukin
Inr initiator
IRAK1, IRAK4 IL-1 receptor associated kinase 1, 4 IκB inhibitor of NFκB (nuclear factor κB) Kan/Neo neomycin phosphotransferase gene lacI repressor of lactose operon
lacO lactose operator
LPS lipopolysaccharide LRR leucin-rich repeat Luc luciferase
MAP mitogen-activated protein
MBD 1-3 methyl CpG binding domain protein 1-3 MBP myelin basic protein
MCS multiple cloning site
MeCP2 methyl CpG binding protein 2 MHC major histocompatibility complex mRNA messenger RNA
MyD88 myeloid differentiation primary-response protein 88 N/P-ratio Molar ratio of PEI nitrogen to DNA phosphate
NFκB nuclear factor 'kappa-light-chain-enhancer' of activated B-cells NIS sodium iodide symporter
NK natural killer cell ODN oligodeoxynucleotide ori origin of replication p/s photons per second
PAMP pathogen-associated molecular pattern PBS phosphate-buffered saline
PCR polymerase chain reaction pDNA plasmid DNA
polyA polyadenylation signal
PSMA prostate specific membrane antigen PSMC porcine smooth muscle cell
QPCR real-time PCR RLU Relative light units RNA ribonucleic acid RNAPol RNA polymerase RSV rous sarcoma virus
S/MAR scaffold matrix attachment region SAF-A scaffold attachment factor A
6 Appendix 66
SV40 simian virus 40
TAB1, TAB2 TAK1-binding protein 1, 2
TAK1 transforming growth-factor-β-activated kinase 1 TH T helper cell
TIR Toll/IL-1 receptor TLR toll-like receptor
TNF-α tumour necrosis factor α
TRAF6 TNF receptor associated factor 6 UBC13 ubiquitin-conjugating enzyme 13
UEV1A ubiquitin-conjugating enzyme E2 variant 1 UTR untranslated region
w/w weight to weight ratio Zeo zeosine resistance gene
6.2.
Publications
6.2.1. Poster presentations
• Terese Magnusson, Verena Russ, Isabella Stapff, Rudolf Haase, Ernst Wagner
and Manfred Ogris: Optimising synthetic ex vivo transferrin mediated transfection of the erythroleukemia cell line K562 with episomal plasmids, DGGT, 2008, Berlin, Germany
• Terese Magnusson, Rudolf Haase, Armin Baiker, Ernst Wagner and Manfred
Ogris: Sustained, high transgene expression in liver with CpG-free plasmids using optimised promoter-enhancer combinations, combined ESGCT and DGGT, 2009, Hannover, Germany.
• Rudolf Haase, Terese Magnusson, Ernst Wagner and Manfred Ogris: Generation
of the novel, synthetic hybrid SCE promoter for gene-therapeutical applications, DGGT, 2010, Munich, Germany.
6.2.2. Publications
• Rudolf Haase, Orestis Argyros, Suet-Ping Wong, Richard P Harbottle, Hans J
Lipps, Manfred Ogris, Terese Magnusson, Maria Vizoso Pinto, Jürgen Haas, Armin Baiker: pEPito: a significantly improved non-viral episomal expression vector for mammalian cells, BMC Biotechnol., 2010, 10-20.
• Sina Rupprecht, Claudia Hagedorn, Davide Seruggia, Terese Magnusson, Ernst
Wagner, Manfred Ogris, Hans J. Lipps: Controlled removal of nonviral episomal vector from transfected cells, Gene, 2010, 466 (1-2): 36-42
• Philipp Dussmann, Judith I. Pagel, Sabina Vogel, Terese Magnusson, Rene
Zimmermann, Ernst Wagner, Wolfgang Schaper, Manfred Ogris, Elisabeth Deindl:
Live in vivo imaging of Egr-1 promoter activity during neonatal development, liver regeneration and wound healing. Submitted.
6 Appendix 68
• Terese Magnusson, Rudolf Haase, Ernst Wagner and Manfred Ogris: Sustained,
high transgene expression in liver with CpG-free plasmids using optimized promoter-enhancer combinations. Submitted.
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